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Oxidative stress and abdominal aortic aneurysm: Potential treatment targets. 1

Theophilus I. Emeto 1,2†, Joseph V. Moxon 1†, Minnie Au 2,3 and Jonathan Golledge 1,4†* 2

1 The Vascular Biology Unit, Queensland Research Centre for Peripheral Vascular Disease, College of 3 Medicine and Dentistry, James Cook University, James Cook Drive, Douglas, Townsville, QLD 4 4811, Australia 5

2 Discipline of Public Health and Tropical medicine, College of Public Health, Medical and 6 Veterinary Studies, James Cook University, Townsville, QLD 4811, Australia 7

3 The Department of Internal Medicine, The Townsville Hospital, Townsville, QLD 4814, Australia; 8

4 The Department of Vascular and Endovascular Surgery, The Townsville Hospital, Townsville, QLD 9 4814, Australia 10

*Author to whom correspondence should be addressed; E-Mail: jonathan.golledge@jcu.edu.au 11 Tel.: +61-747961417; Fax: +61-7479614 12

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† These authors contributed equally. 15

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Running Title – Emeto- Oxidative stress and abdominal aortic aneurysm 17

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mailto:jonathan.golledge@jcu.edu.au

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Abstract 34

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Abdominal aortic aneurysm (AAA) is a significant cause of mortality in older adults. A key 36 mechanism implicated in AAA pathogenesis is inflammation and the associated production of 37 reactive oxygen species (ROS) and oxidative stress. These have been suggested to promote 38 degradation of the extracellular matrix and vascular smooth muscle apoptosis. Experimental and 39 human association studies suggest that ROS can be favourably modified to limit AAA formation and 40 progression. In this article, we discuss mechanisms potentially linking ROS to AAA pathogenesis and 41 highlight potential treatment strategies targeting ROS. Currently, none of these strategies have been 42 shown to be effective in clinical practice. 43

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Key words- abdominal aortic aneurysm, pharmacotherapy, oxidative stress, clinical trial, and animal 47 models 48

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Introduction 67

Abdominal aortic aneurysm (AAA) is a degenerative disease of the aorta common in people aged > 68

65 years.(1, 2) AAA is usually asymptomatic until rupture which is frequently fatal.(1-4) However, 69

rupture of small AAAs is rare.(5) The main focus of AAA management is to prevent rupture, 70

therefore current guidelines recommend that patients with large AAAs (>5-5.5cm) undergo 71

endovascular stent graft or open surgical repair.(4, 6) Patients with small AAAs undergo regular 72

imaging to monitor AAA diameter, although up to 70% of these individuals eventually require AAA 73

repair.(2) 74

AAA is believed to result from an aberrant interaction between genetic factors and the environment 75

which aggravates the normal ageing processes.(1, 2) One of the key features of AAA is vascular wall 76

inflammation associated with significant production of reactive oxygen species (ROS).(3, 6) ROS, 77

which include hydrogen peroxide (H2O2), superoxide (O2−), and hydroxyl radical (•OH) are oxygen-78

derived chemical molecules with high reactivity.(7, 8) Reports from recent clinical and experimental 79

investigations suggest that oxidative stress, i.e. production of ROS in excess of antioxidant protection, 80

is involved in the vascular degeneration found in AAA.(9-11) Imbalance in the activity of endogenous 81

pro-oxidative enzymes such as nicotinamide adenine dinucleotide phosphate oxidase (NOX) and 82

xanthine oxidase (XO), the mitochondrial respiratory chain and the antioxidant enzymes such as 83

glutathione peroxidase, heme oxygenase (HO), superoxide dismutase (SOD), thioredoxin (TRX), and 84

catalase results in excessive ROS.(12, 13) This is implicated in vascular cell dysfunction, lipid and 85

protein peroxidation, and deoxyribonucleic acid (DNA) damage which can result in permanent 86

cellular damage and death.(7, 12, 13) ROS regulate the degradation and remodelling of the 87

extracellular matrix (ECM) by upregulating proteolytic enzymes such as matrix metalloproteinases 88

(MMPs), (14) activating signalling kinases and transcription factors such as nuclear factor kappa beta 89

(NF-κB) and activator protein 1 (AP-1),(15-17) and promoting vascular smooth muscle cell (VSMC) 90

apoptosis.(18, 19) ROS also regulate fibroblast proliferation and macrophage and mononuclear 91

lymphocyte infiltration.(7, 20, 21) 92

In this article we discuss mechanisms potentially linking ROS to AAA pathogenesis and review 93

potential treatment strategies targeting ROS in the management of AAA based on experimental and 94

human studies. 95

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Literature Search 97

A literature search was conducted to identify studies assessing antioxidants as therapeutic targets for 98

AAA using the Embase (1980), MEDLINE (1966), SCOPUS (1996), Web of Science (1965), and 99

Cochrane Library databases (1992) from inception to the 25th of May, 2015. The following search 100

terms were applied either as single or combined searches: “abdominal aortic aneurysm” OR “AAA”, 101

[Title/Abstract] AND “antioxidant treatment” OR “antioxidant targets” OR “antioxidant therapy”, 102

AND/OR “clinical studies” OR “human studies”, AND/OR “animal studies” OR “experimental 103

studies”. Abstracts were analysed for relevance and studies describing the therapeutic potential of 104

antioxidants in AAA pathogenesis were retrieved. Human and animal studies investigating the 105

therapeutic potential of antioxidants in AAA were included. Included article references were hand 106

searched for relevant publications meeting the inclusion criteria. Studies excluded include those in 107

languages other than English and studies unrelated to oxidative stress. 108

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Evidence linking ROS with AAA 110

ROS such as NO and O2− are produced by all vascular cell types principally via membrane-associated-111

specific enzymes such as NOX, XO, and nitric oxide synthase (NOS). (7, 13) Both animal and human 112

studies have associated elevated concentrations of ROS, or surrogate markers of ROS with AAA. (11, 113

22-24) 114

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Animal studies 116

Evidence from different animal models implicates ROS in AAA pathogenesis. Nakahashi and 117

colleagues reported that HO-1 expression was significantly upregulated within the elastase-induced 118

rat model of AAA. (24) They further demonstrated the localisation of HO-1 to infiltrative 119

macrophages within the aneurysmal aortic wall. Similarly, iNOS produced NO has been reported to 120

exacerbate experimental AAA development.(25) Lizarbe et al. reported a reduction in aortic diameter 121

and MMP-13 expression in mice administered the iNOS inhibitior, 1400 W.(14) In the angiotensin II 122

(AngII)-induced mouse model of AAA, 8-isoprostane,(23) and 8-hydroxy-2' -deoxyguanosine (8-123

OHdG), (9) concentrations (markers of oxidative stress) have been reported to be high within the 124

aneurysmal aorta. Similarly, high 8-OHdG content and glutathione peroxidase expression were 125

reported within the calcium chloride (CaCl2)-induced mouse model of AAA. (26) Infiltrating 126

inflammatory cells and their products have been reported to be involved in aortic wall degradation and 127

ensuing AAA formation.(3, 27) (28) 128

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Human associated studies 130

Dubick and colleagues reported low levels of vitamin C, and lower copper-zinc SOD (CuZnSOD) 131

activity in aortic tissue samples from 29 patients with AAA and 14 patients with atherosclerotic 132

occlusive disease (AOD) compared to non-diseased control aortas.(29) SOD is a family of 133

metalloenzymes including CuZnSOD, manganese SOD (MnSOD), and extracellular SOD (EcSOD) 134

that catalyses the dismutation of O2− into oxygen and H2O2 thereby maintaining vascular NO 135

concentrations.(30, 31) High MnSOD and low CuZnSOD activities were observed in a study 136

evaluating the effect of hypertension on aortic antioxidant status in human AAA and AOD.(42) In 137

contrast, a separate study suggested that both CuZnSOD and MnSOD activities were lower in infra-138

renal aortic biopsies collected from patients with intact and ruptured AAAs compared to non-139

aneurysmal organ donors.(32) This study also suggested that glutathione reductase and glutathione 140

peroxidase were downregulated whilst lipid peroxidation products were high in AAA patients, 141

particularly those with ruptured aneurysms, compared to controls.(32) Aberrant lipid peroxidation has 142

been associated with aortic cell apoptosis and necrosis, (discussed in (33, 34)) which potentially 143

promotes aortic weakening and AAA. Evidence also suggests that dysregulated antioxidant protective 144

mechanisms,(35-42) and/or low levels of exogenous antioxidants (vitamin C, vitamin E) can 145

exacerbate ROS activity.(7, 13, 43) In addition, Miller et al. reported high expression of O2− and NOX 146

activity in biopsies of human aneurysmal aortas compared with biopsies from adjacent non-147

aneurysmal aortas.(11) Zhang et al. reported high iNOS expression in the media and adventitia of 148

human AAA tissues compared to controls.(44) They suggested that iNOS promotes AAA progression 149

by selectively stimulating the formation and activity of the NO-derived oxidant peroxynitrite (ONOO-150

) with consequent aortic oxidative injury and AAA.(44) 151

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Collectively, these studies suggest that oxidative stress is associated with the presence of AAA. It 152

remains to be shown whether targeting ROS or their modulators would be an effective strategy in 153

managing AAA. 154

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Potential mechanisms linking oxidative stress with AAA and associated therapeutic targets 156

Sies defined oxidative stress as an imbalance between ROS and antioxidants in favour of the oxidants 157

leading to cellular injury and damage.(45) There is a close association between oxidative stress and 158

inflammation as evidence suggests that oxidative stress induces inflammation, which in turn 159

potentiates oxidative stress with resultant injury to tissues.(11, 46, 47) The human body is equipped to 160

maintain the oxidant-antioxidant balance to avoid injury in physiological conditions, but potentially 161

not in pathophysiological conditions like AAA (discussed in (45, 48)). 162

A number of endogenous enzyme systems regulating oxidative stress in AAA have been researched in 163

both pre-clinical and clinical studies in the past decade (Figure 1). The following sections summarise 164

the potential mechanisms linking these pathways to AAA and strategies which potentially, may be 165

used therapeutically to limit AAA (Figure 2). 166

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Xanthine and NADPH oxidases 168

NOX activity has been reported to be markedly elevated by stimuli thought to be key in the 169

pathogenesis of AAA such as Ang II, TNF-α, mechanical stretch, and endothelin-1 acting both 170

through transcriptional pathways and posttranslational modification of oxidase regulatory subunits.(7, 171

49) Ang II for example, has been reported to induce mitochondrial dysfunction via a protein kinase 172

C–dependent pathway by activating NOX and forming NO, ONOO-, and O2− (Figure 2).(50) 173

Oxidative stress has been reported to enhance expression of angiotensin converting enzyme (ACE) 174

which is responsible for the conversion of angiotensin I (Ang I) to Ang II.(51) In addition, mice 175

overexpressing the NOX catalytic subunit Nox1 within VSMC are reported to have an enhanced 176

response to Ang II, exhibit increased O2− and H2O2 production and have aortic thickening.(52, 53) 177

Thomas et al. employing the Ang II-infused mouse model of AAA reported that deletion of p47phox, 178

a cytosolic subunit of NOX, abrogated NOX activity, reduced production of ROS within the aorta and 179

within infiltrating leukocytes, and significantly reduced the incidence and progression of AAA.(54) In 180

contrast, inhibition of Nox1 has been associated with resistance to aortic dissection and AAA 181

development in mouse models.(55) However, a deficiency in another NOX catalytic subunit, Nox2 182

was reported to exacerbate Ang II-induced AAA in mice and promote interleukin-1 beta (IL-1β) and 183

MMP-9 expression.(56) Inhibition of IL-1β or IL-1β receptor expression has been reported to confer 184

AAA resistance in mouse models.(57) 185

XO is also a H2O2 and O2− generating enzyme formed from the proteolytic breakdown of xanthine 186

dehydrogenase.(13) Experimental evidence suggests XO is expressed by aortic endothelial cells in a 187

NOX dependent manner,(58) and is actively involved in lipid peroxidation and damage to the ECM 188

(discussed in (59)). However there are limited studies investigating the specific role of XO in AAA 189

pathogenesis. 190

Taken together, these studies suggest a potential mechanism by which NOX may modulate AAA 191

pathogenesis via effects on the renin-angiotensin system (RAS), inflammatory cytokines and MMPs. 192

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Zhang et al. reported that cilostazol, a phosphodiesterase III inhibitor that selectively targets cyclic 193

adenosine monophosphate (cAMP), inhibited AAA development in an elastase-induced rat model. 194

(60) Cilostazol is a 2-oxo-quinoline derivative with antithrombotic, vasodilator, antimitogenic and 195

cardiotonic properties and is indicated for intermittent claudication in patients with peripheral arterial 196

disease.(61) Cilostazol was shown to significantly reduce NOX activity and ROS concentration with 197

consequent inhibition of MMP-2 and MMP-9 expression, and NF-κB activation (P < 0.05).(60) In a 198

CaCl2 mouse model of AAA, mice orally administered apocynin (a NOX inhibitor) were shown to 199

have reduced MMP-2, MMP-9 and NO metabolite (NO2 and NO3) expression within aortic tissues, 200

and decreased aneurysm formation compared to controls.(18) In contrast, Kigawa et al. reported that 201

mice deficient in NOX demonstrated increased incidence of AAA, albeit with decreased ROS 202

expression, within an Ang II-induced low-density lipoprotein receptor knock-out (Ldlr-/-) mouse 203

model of AAA.(56) Collectively, these studies suggest that medication targeting both NOX and XO 204

pathways may limit AAA formation and progression, although more studies are needed to validate 205

these findings. 206

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Lipoxygenases 208

Lipoxygenases are non-heme iron–containing enzymes that catalyse the stereospecific deoxygenation 209

of polyunsaturated fatty acids with a 1, 4-cis, cis-pentadiene structure (62, 63) and are also proficient 210

in the generation of ROS, or augmenting leukocyte ROS production.(8, 64) Lipoxygenases are 211

required for the biosynthesis of leukotrienes, which are key inflammatory and chemotactic 212

mediators.(65) For example, inhibition of lipoxygenases including 5- lipoxygenase (5-LO) has been 213

reported to reduce the expression of MMP-2 and -9, and phagocytic macrophage infiltration.(66-68) 214

The proteolytic breakdown of the ECM by MMPs has been reported to be one of the key mechanism 215

involved in AAA pathogenesis (for a detailed review of MMPs and AAA, please see (69)). It has been 216

suggested that by promoting inflammation and the proteolytic degradation of the ECM via effects on 217

MMPs expression and recruitment of inflammatory cells, lipoxygenases may play a role in inducing 218

AAA formation. 219

A growing body of evidence has implicated lipoxygenases in the pathogenesis of AAA as illustrated 220

in Table 1.(46, 66, 67) A recent study by Bhamidipati et al. using an elastase perfused- and an Ang II-221

infused Ldlr-/- mouse models of AAA demonstrated that 5- lipoxygenase (5-LO) inhibition attenuated 222

AAA formation and progression.(67) The authors reported that genetic deletion of 5-LO within the 223

elastase infused mouse model resulted in a 71% reduction in aortic dilatation compared with wild-224

type controls. Mice administered 30 mg/kg/day of AZD4407 (a selective 5-LO inhibitor) were also 225

shown to be resistant to AAA formation and progression associated with decreased MMP-9 enzymatic 226

activity and immune cell infiltration within aortic tissue. These findings were further strengthened 227

through studies within the Ang II-infused Ldlr-/-mouse model of AAA, were they reported that 228

administration of AZD4407 within the chow for 28 days inhibited 5-LO activity within the circulation 229

resulting in a 54% reduction in aneurysm formation compared to control.(67) 230

Edaravone (3-methyl-1-phenyl-2-pyrazoline-5-one), a powerful antioxidant indicated as a therapeutic 231

agent for acute cerebral infarction,(70) was reported to significantly reduce the expression of MMP-2, 232

MMP-9 and ROS, and abrogate AAA formation and expansion in the combined elastase-induced and 233

CaCl2-induced rat model of AAA.(71) In addition, earlier studies by Zhao et al. found that ApoE–/– 234

mice lacking 5-LO genes and fed a cholic acid-rich diet demonstrated a reduced incidence of 235

AAA.(66) Consistent with this, Ang II-infused ApoE–/– mice deficient in leukotriene B4 receptor 1 236

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(BLT1) were shown to exhibit decreased incidence and size of AAA.(72) In a similar model, a BLT 237

antagonist (CP-105,696) limited AAA formation when administered at the same time as Ang II 238

infusion, but did not limit progression of established aneurysms. (68). Cao et al. employing the Ang 239

II-infused ApoE–/– mouse model of AAA, reported that mice deficient in 5-LO or administered a 5-LO 240

activating protein (FLAP) inhibitor (MK-0591) had a similar incidence of AAA development to 241

controls.(73) 242

A human association study reported higher expressions of leukotriene C4 synthase (LTC4S), 5-LO 243

and FLAP within aneurysmal aortas compared to non-aneurysmal donor aortas.(74) Others have also 244

reported high production of leukotriene B4 (LTB4) by polymorphonuclear leukocytes localised within 245

intra-luminal thrombus samples collected from patients undergoing aneurysm repair.(75) A study 246

involving 613 men aged ≥65 with AAA and 707 randomly selected age-matched controls failed to 247

find an association between 7 single nucleotide polymorphisms in ALOX5AP, the gene encoding 248

FLAP, with human AAA.(76) Together, these reports fail to show a consistent role of lipoxygenase in 249

human and experimental AAA. 250

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Cyclooxygenases 252

Cyclooxygenases (COX) are the key enzymes involved in the conversion of arachidonic acid to 253

prostaglandins.(77) It has been reported that COX-2 and its metabolite, prostaglandin E2 (PGE2), are 254

highly expressed within aneurysmal tissue and exacerbate VSMC apoptosis.(78-80) COX-2 255

expression was also reported to be associated with increased macrophage infiltration,(81) and 256

increased MMP-2 expression and aortic remodelling. Collectively, this information suggests that 257

COX-2 may play a significant role in AAA pathogenesis via effects on VSMC integrity and 258

inflammation, stimulating interest in treatments targeting the COX pathway as a means of limiting 259

AAA. Celecoxib, a nonsteroidal anti-inflammatory drug (NSAID), and a selective COX-2 inhibitor, 260

was reported to reduce the incidence and severity of AAA within the Ang II-induced mouse model in 261

both hyperlipidaemic and nonhyperlipidaemic mice.(82) In the same mouse model of AAA, Gitlin et 262

al. reported that mice with genetic deletion of COX-2 failed to develop AAA. They reported a 73% 263

and 90% reduction in AAA incidence following 7 and 21 days of Ang II infusion respectively and a 264

marked decrease in the aortic expression of the macrophage marker CD68, MCP-1 and macrophage 265

inflammatory protein-1alpha (MIP-1α).(81) This is in contrast to the study by Cao et al. reporting that 266

genetic deletion of COX-2 had no significant effect on AAA development within the Ang II-induced 267

AAA mouse model.(83) However, two recent studies provide evidence that mice administered 268

celecoxib have decreased AAA progression and rupture within the Ang II infused mouse model of 269

AAA.(84, 85) Another selective COX-2 inhibitor, MF-tricyclic, was reported to inhibit MMP-9 270

expression and consequently AAA growth within an elastase-induced rat model of AAA.(86) In 271

support, two other studies employing the same elastase-induced rat model of AAA reported that 272

indomethacin (a non- selective COX-2 inhibitor) significantly inhibited PGE2 and MMP-9 expression 273

thereby preserving elastin integrity and reducing AAA expansion with no effect on the inflammatory 274

infiltrate.(87, 88) Conversely, Armstrong and colleagues reported that indomethacin and rofecoxib (a 275

selective COX-2 inhibitor) failed to impede aneurysm expansion within the elastase-induced AAA rat 276

model.(89) Furthermore, in a small case-control study involving 15 subjects on NSAIDs and 63 277

patients without NSAID, it was reported that AAA growth was significantly reduced in patients 278

receiving NSAIDs compared to controls without NSAIDs.(80) COX inhibitors have been associated 279

with increased incidence of cardiovascular events (myocardial infarction stroke and cardiovascular 280

death), and it is therefore unclear whether they would be safe to give patients with AAA.(90, 91) The 281

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conflicting evidence from experimental studies regarding their therapeutic potential does not currently 282

support their trialling in AAA patients. A summary of studies investigating the role of COX in AAA 283

pathogenesis is given in Table 2. 284

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Nitric oxide synthases 286

The family of NOS including inducible NOS (iNOS), endothelial NOS (eNOS) and neuronal NOS 287

(nNOS) are capable of generating NO and L-citrulline by catalysing the oxidation of L-arginine. Both 288

eNOS and nNOS are selectively expressed whereas iNOS is widely distributed within multiple cell 289

types.(12, 92) NO is an important regulator of cardiovascular homeostasis, however evidence suggest 290

that under specific conditions, exaggerated production of NO by iNOS may lead to tissue damage 291

through several mechanisms including the formation of reactive nitrogen species.(93) In addition, 292

uncoupling of eNOS from its cofactors is associated with increased generation of O2−,(93, 94) and 293

consequent tissue damage which has been suggested to be relevant in AAA pathogenesis.(94, 95) 294

A number of medications which were not developed to modulate NOS have been suggested to have 295

pleiotropic effects on NOS which in part explain their ability to limit AAA within experimental 296

models (Table 3). For example a number of studies suggest that statin therapy may limit AAA 297

development due to its antioxidant and anti-inflammatory effects.(96-102) In an elastase induced rat 298

model of AAA, Kalyanasundaram et al. reported that simvastatin administration resulted in the 299

downregulation of several oxidative stress-related genes including SOD2, HO-1, NOS3 and 300

thioredoxin reductase 1 (TRXR1) with consequent AAA inhibition.(96) Simvastatin was also shown 301

to reduce NF-κB, MMP-9 and MMP3 expression.(96) This is supported by the study by Steinmetz et 302

al. which demonstrated that simvastatin inhibited AAA formation independent of serum cholesterol 303

levels in the C57BL/6 wild type and the ApoE-/- mice using the elastase-infused mice model of 304

AAA.(97) They reported a simvastatin-mediated decrease in MMP-9 and increase in TIMP-1 305

expression, but did not measure any ROS.(97) In contrast, others have reported that simvastatin had 306

no significant effect on aortic diameter within the Ang II-induced mouse model of AAA.(103) In two 307

previous studies, fenofibrate, a peroxisome proliferator-activated receptor alpha (PPARα) activator 308

used clinically to reduce triglycerides, was shown to abrogate Ang II-induced AAA within Ldlr-/- and 309

ApoE-/- mice.(104, 105) Fenofibrate upregulated eNOS within the tunica intima and iNOS within the 310

tunica media and adventitia associated with reduced Ang II induced AAA formation.(105) In addition, 311

within a CaCl2 mouse model of AAA, iNOS deficient mice were shown to be partly resistant to AAA 312

formation associated with decreased MMP-2, MMP-9 and NO expression within aortic tissues.(18) 313

In contrast, Gao et al. reported that mice deficient in the eNOS cofactor tetrahydrobiopterin (H4B) 314

were prone to Ang II-induced AAA formation.(94) They reported that oral folic acid treatment 315

upregulates the expression and activity of the H4B salvage enzyme dihydrofolate reductase within the 316

endothelium, restores eNOS function and inhibited AAA formation.(94) In a further study employing 317

the Ang II infused ApoE-/- mice model of AAA, Siu and colleagues also demonstrated that oral 318

administration of folic acid limited aortic elastin degradation, macrophage infiltration and 319

significantly inhibited AAA expansion by upregulating eNOS activity.(95) Similarly, deletion of the 320

eNOS gene within ApoE-/- mice has been associated with increased AAA formation.(106) These 321

studies suggest that due to the complexity of these enzymes, the underlying difference in the animal 322

models involved, and the inconsistent findings it is currently unclear how NOS regulation is involved 323

in AAA pathogenesis. 324

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Heme oxygenases and thioredoxin 326

HO-1 is an enzyme that reduces oxidative stress by catalysing heme to carbon monoxide, biliverdin, 327

bilirubin, and free ferrous iron.(12, 13, 107, 108) The endogenously produced carbon monoxide is 328

reported to serve as a second messenger influencing cellular proliferation, inflammation, and 329

apoptosis.(109) 330

Nakahashi et al. were the first to suggest that HO-1 may be beneficial in limiting AAA 331

development.(24) The authors conducted a microarray analysis to investigate aortic gene expression 332

following a femoral arteriovenous fistula to increase laminar shear and wall strain within an elastase-333

induced rat model of AAA. They found that flow loading decreased AAA diameter and upregulated 334

HO-1 expression.(24) 335

Two contradictory case-control studies were found examining the relationship between the HO-1 gene 336

promoter region and AAA prevalence in humans.(110, 111) Short (

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Catalase is an enzyme responsible for the degradation of H2O2 to water and oxygen and has 368

cardiovascular protective effects,(117) including inhibition of Ang II-induced aortic thickening.(37) 369

Patients with AAA have been reported to have low catalase expression within circulating neutrophils 370

and plasma.(118) Catalase has been reported to abrogate the H2O2-mediated increases in MMP 371

expression and inflammatory cytokine secretion. (117, 119) 372

Several studies have investigated the relationship between SOD, catalases and AAA as shown in 373

Table 4.(119-124) In a study, employing an elastase-induced rat model of AAA, Sinha et al. reported 374

a significant increase in MnSOD levels and MMP-9 activity in aneurysmal aortas during the early 375

stages of aneurysm formation. CuZnSOD and EcSOD levels were similar in AAAs and control 376

aortas.(120) The authors reported that a SOD mimetic, TEMPOL, increased MMP-2 and MMP-9 377

activity by more than 2-fold in aortic explant cultures, and suggested that strategies aimed at 378

inhibiting oxidative stress during AAA formation should focus on MnSOD.(120) In a recent study 379

employing the CaCl2 induced mouse model of AAA, a standardised extract from Ginkgo biloba, EGb 380

761, reported to possess antioxidant and free radical scavenging properties,(125) was shown to 381

significantly reduce NF-κβ protein expression, MMP-2 and MMP-9 activities within the aorta, inhibit 382

SOD and catalase activity, and decrease aneurysm size.(126) Other studies however report that 383

increased SOD is beneficial against AAA formation.(121) For example, another natural product, 384

diferuloylmethane (curcumin), a phenol found in the dietary spice turmeric,(127) has been reported to 385

exert anti-inflammatory effects via inhibition of ROS production and enhanced SOD expression.(121) 386

Curcumin was also reported to inhibit aneurysm formation.(128) An initial study using the elastase-387

induced mouse model of AAA showed that curcumin inhibited AAA formation by decreasing aortic 388

inflammation and MMP-9 expression.(129) Similarly, in the Ang II-induced mouse model of AAA, 389

Hao et al. reported that oral administration of curcumin significantly decreased aortic macrophage 390

infiltration, MCP-1 and TNF-α concentrations and AAA incidence, and increased SOD activity.(121) 391

Cobalt chloride (CoCl2), an inhibitor of the hypoxia-inducible factor alpha (HIF-1α) degrading prolyl 392

hydroxylase domain protein,(130) was reported to restore SOD and catalase expression within a 393

CaCl2 model of AAA, and inhibit NF-κβ phosphorylation, cytokine expression and consequent AAA 394

formation.(123) Taken together, there is no consistent evidence on the role of SOD in AAA 395

pathogenesis. 396

A study examining the effect of tamoxifen, a selective oestrogen receptor modulator with potent 397

antioxidant and vasodilator properties,(131) demonstrated that rats receiving 10mg/kg/day of 398

tamoxifen subcutaneously exhibited a marked increase in aortic catalase expression associated with 399

significant inhibition of neutrophil infiltration and AAA expansion compared to controls within the 400

elastase-induced rat model of AAA.(124) The direct irreversible catalase inhibitor 3-amino-1, 2, 4-401

triazole (AT) was shown to significantly abrogate the aneurysm inhibitory effect of tamoxifen by 402

roughly 30%.(124) In addition, transgenic ApoE-/- mice overexpressing the human catalase gene 403

within VSMC where shown to be resistant to Ang II induced aortic wall remodelling thought to be 404

involved in early AAA formation.(132) This finding was supported by a similar study within the 405

CaCl2 induced mice model of AAA, where both transgenic mice over expressing catalase and mice 406

administered polyethylene glycol-catalase (PEG-catalase) where shown to exhibit decreased MMP 407

activity, decreased VSMC apoptosis and decreased AAA formation.(119) However, mice transgenic 408

for catalase did not have altered H2O2 concentrations.(119) These studies suggest that upregulating 409

aortic catalase activity is a putative mechanism to limit AAA progression. 410

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Studies investigating exogenous and phenolic antioxidants 413

Exogenous antioxidants including folic acid, vitamin C (ascorbic acid), and vitamin E (α-Tocopherol) 414

are reported to modulate ROS production with implications for AAA pathogenesis.(23, 133-136) For 415

example, vitamin E which has been reported to also inhibit COX expression,(133) has been shown to 416

inhibit AAA formation in two different rodent models of AAA.(23, 24, 137) In the Ang II-induced 417

ApoE-/- mice model of AAA, Gavrila et al. reported decreased concentrations of markers of oxidative 418

stress and aortic macrophage infiltration along with reduction in maximum AAA diameter and 419

incidence of rupture in mice receiving vitamin E.(23) Gopal et al. reported that dietary 420

supplementation with vitamin E and β-carotene confers substantial protection against Ang II induced 421

AAA formation. (137) In an elastase-induced rat model of AAA, Nakahashi and colleagues reported 422

that rats receiving vitamin E had significantly decreased AAA expansion compared to controls.(24) 423

Unfortunately in a randomised double-blind placebo-controlled trial, Tornwall et al. failed to show 424

any significant association between vitamin E supplementation and the incidence of AAA.(138) The 425

latter study did not include aortic imaging and simply relied on the clinical presentation of AAA. This 426

study does not therefore rule out a benefit of vitamin E in limiting AAA growth. 427

Vitamin C is considered to be one of the most efficient water-soluble antioxidant in human plasma 428

with the ability to scavenge ROS, regenerate α-tocopherol and reduce H4B preventing eNOS 429

uncoupling.(12, 43, 139) Shang et al. in a study using the elastase-induced rat model of AAA 430

demonstrated that daily intraperitoneal injection of vitamin C (100mg/ml) significantly decreased 8-431

hydroxylguanine and 8-isoprostane (markers of oxidative stress), MMP-2, MMP-9 and IL-1β 432

expression, upregulated TIMP-1 and -2 expression and abrogated AAA expansion by 26%.(10) In a 433

similar study employing the combined elastase and CaCl2-induced rat model, vitamin C 434

administration was shown to limit AAA formation through downregulation of MMP-9, MCP-1, IL-435

1β, TNFα, CD68, and ROS (8-hydroxylguanine).(140) 436

Resveratrol (3,5,4′-trihydroxy-trans-stilbene), a dietary polyphenol commonly found in red wine and 437

grape skin with antioxidant,(141-144) anti-inflammatory,(145) and anti-angiogenic(146) effects has 438

been reported to inhibit experimental AAA.(26, 147) In a CaCl2 induced mice model of AAA, 439

resveratrol (at 100mg/kg/day) was reported to prevent AAA development by attenuating the 440

expression of glutathione peroxidase, MCP-1, TNF-α and CD68 and reducing the activity of MMP-9 441

and -2.(26) One limitation of this study was the effect of resveratrol at other doses or time points was 442

not reported especially since 100mg/kg/day is quite a large dose within mice. Palmieri et al. studied 443

the effect of resveratrol within the elastase-induced rat model of AAA.(147) They reported that male 444

Sprague-Dawley rats receiving 10 mg/kg/day resveratrol one week before until two weeks following 445

AAA induction exhibited significantly decreased CD62L-monocyte subset expansion, CD143 446

monocyte expression, TNFα expression and MMP-9 activity with consequent inhibition of AAA 447

development.(147) In addition, the polyphenolic flavonoid quercetin, which has antioxidant and anti-448

inflammatory properties,(148) was reported to reduce the aortic expression of MMP-2, MMP-9, and 449

cathepsin, and limit macrophage infiltration with consequent decrease in AAA diameter.(149) In a 450

second experiment, the authors reported the inhibitory effects of quercetin on AAA incidence via 451

attenuation of oxidative stress and MMP activation was in part through the modulation of c‑Jun N‑452

terminal kinase (JNK)/AP‑1 signalling.(150) 453

454

455

12

The potential of modulating oxidative stress to limit AAA growth in patients 456

The studies noted above provide good evidence that markers of oxidative stress are upregulated in 457

experimental and human AAA. The wide range of oxidative stress pathways upregulated and the 458

number of interactions between these makes it extremely complex to design interventions which can 459

effectively reduce aortic oxidate stress long-term (see Figure 2). This is particularly difficult where 460

high levels of oxidative stress are already present such as within established AAAs. Many of the 461

interventions which have been successful in experimental models have been applied prior to or at the 462

time of AAA induction. However, treatments are needed for established AAAs in patients.(151) 463

Recently a number of strategies which were successfully applied in animal models, such as 464

doxycycline mediated MMP inhibition and use of a mast cell inhibitor, have been reported to be 465

ineffective at limiting AAA growth in patients.(152, 153) It is currently not clear why previous 466

promising strategies to limit AAA growth have not been translated to patients, although suggested 467

reasons include poor design of previous animal studies, inappropriate animal models and ineffective 468

approaches to inhibiting the pathways that were targeted. Given that patients with AAA usually have 469

multiple co-morbidities any treatment to limit AAA growth would need to be very safe. Based on the 470

animal data presented in this review a number of non-toxic antioxidant approaches have potential to 471

limit AAA, such as vitamin E, vitamin C and polyphenolics (curcumin, quercetin, resveratrol). 472

Despite the promising experimental studies there are no clinical trial data to suggest that such simple 473

antioxidant therapies are effective. For example, a clinical trial failed to show any significant 474

association between vitamin E supplement and the incidence of AAA, although no aortic imaging was 475

included in this trial.(138) Whether such simple approaches to modifying oxidative stress can achieve 476

a sustained reduction in aortic oxidative stress is uncertain. Clinical trials to examine the value of 477

these agents in established AAAs are needed to answer this. 478

479

480

481

482

483

484

485

486

487

488

489

490

491

492

13

List of abbreviations 493

5-LO 5-lipooxygenase

AAA Abdominal aortic aneurysm

Ang II angiotensin II

AP-1 Activator protein-1

ApoE-/- apolipoprotein E deficient

CaCl2 Calcium chloride

cAMP cyclic adenosine monophosphate

CoCl2 Cobalt chloride

COX Cyclooxygenase

CuZnSOD Copper-Zinc SOD

ECM Extracellular matrix

EcSOD Extracellular SOD

eNOS Endothelial NOS

FLAP 5-lipooxygenase activating protein

H4B Tetrahydrobiopterin

HMG-CoA 3-hydroxyl-3-methylglutaryl coenzyme A

HO Heme oxygenase

IL-1β interleukin-1β

iNOS Inducible NOS

JNK c-Jun N-terminal kinase

MCP-1 Monocyte chemoattractant protein-1

MIP Macrophage inflammatory protein

MMP Matrix metalloproteinase

MnSOD Manganese SOD

14

NADPH Nicotinamide adenine dinucleotide phosphate

NF-κB nuclear factor-κB

nNOS Neuronal NOS

NO Nitric oxide

NOS Nitric oxide synthase

NOX Nicotinamide adenine dinucleotide phosphate oxidase

NSAID nonsteroidal anti-inflammatory drug

PGE2 Prostaglandin E2

ROS Reactive oxygen species

SOD Superoxide dismutase

TIMP-1 tissue inhibitor of metalloproteinase-1

TNFα Tumor necrosis factor α

TNF-α tumor necrosis factor-α

TRX Thioredoxin

VSMC Vascular smooth muscle cells

XO Xanthine oxidase

494

495

Acknowledgments 496

Research undertaken by J.G is supported by the National Health and Medical Research Council 497

(project grant numbers 1063476, 1022752, 1021416, 1020955, 1003707; Practitioner Fellowship 498

number 1019921; and a Centre of Research Excellence number 1000967), the Queensland 499

Government and The Townsville Hospital Private Practice Trust Fund. The funding bodies played no 500

role in the production of this paper. 501

Conflicts of Interest 502

The authors declare no conflict of interest. 503

504

505

15

References 506

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96. Kalyanasundaram A, Elmore JR, Manazer JR, Golden A, Franklin DP, Galt SW, et al. 755 Simvastatin suppresses experimental aortic aneurysm expansion. Journal of vascular surgery. 756 2006;43(1):117-24. 757 97. Steinmetz EF, Buckley C, Shames ML, Ennis TL, Vanvickle-Chavez SJ, Mao D, et al. Treatment 758 with simvastatin suppresses the development of experimental abdominal aortic aneurysms in 759 normal and hypercholesterolemic mice. Ann Surg. 2005;241(1):92-101. 760 98. Schouten O, van Laanen JH, Boersma E, Vidakovic R, Feringa HH, Dunkelgrun M, et al. Statins 761 are associated with a reduced infrarenal abdominal aortic aneurysm growth. Eur J Vasc Endovasc 762 Surg. 2006;32(1):21-6. 763 99. Sukhija R, Aronow WS, Sandhu R, Kakar P, Babu S. Mortality and size of abdominal aortic 764 aneurysm at long-term follow-up of patients not treated surgically and treated with and without 765 statins. Am J Cardiol. 2006;97(2):279-80. 766 100. Evans J, Powell JT, Schwalbe E, Loftus IM, Thompson MM. Simvastatin attenuates the activity 767 of matrix metalloprotease-9 in aneurysmal aortic tissue. Eur J Vasc Endovasc Surg. 2007;34(3):302-3. 768 101. Zhang Y, Naggar JC, Welzig CM, Beasley D, Moulton KS, Park HJ, et al. Simvastatin inhibits 769 angiotensin II-induced abdominal aortic aneurysm formation in apolipoprotein E-knockout mice: 770 possible role of ERK. Arterioscler Thromb Vasc Biol. 2009;29(11):1764-71. 771 102. Shiraya S, Miyake T, Aoki M, Yoshikazu F, Ohgi S, Nishimura M, et al. Inhibition of 772 development of experimental aortic abdominal aneurysm in rat model by atorvastatin through 773 inhibition of macrophage migration. Atherosclerosis. 2009;202(1):34-40. 774 103. Golledge J, Cullen B, Moran C, Rush C. Efficacy of simvastatin in reducing aortic dilatation in 775 mouse models of abdominal aortic aneurysm. Cardiovasc Drugs Ther. 2010;24(5-6):373-8. 776 104. Golledge J, Cullen B, Rush C, Moran CS, Secomb E, Wood F, et al. Peroxisome proliferator-777 activated receptor ligands reduce aortic dilatation in a mouse model of aortic aneurysm. 778 Atherosclerosis. 2010;210(1):51-6. 779 105. Krishna SM, Seto SW, Moxon JV, Rush C, Walker PJ, Norman PE, et al. Fenofibrate increases 780 high-density lipoprotein and sphingosine 1 phosphate concentrations limiting abdominal aortic 781 aneurysm progression in a mouse model. Am J Pathol. 2012;181(2):706-18. 782 106. Kuhlencordt PJ, Gyurko R, Han F, Scherrer-Crosbie M, Aretz TH, Hajjar R, et al. Accelerated 783 atherosclerosis, aortic aneurysm formation, and ischemic heart disease in apolipoprotein 784 E/endothelial nitric oxide synthase double-knockout mice. Circulation. 2001;104(4):448-54. 785 107. Miller FJ, Jr. Aortic aneurysms: It's all about the stress. Arterioscler Thromb Vasc Biol. 786 2002;22(12):1948-9. 787 108. Soares MP, Bach FH. Heme oxygenase-1: from biology to therapeutic potential. Trends Mol 788 Med. 2009;15(2):50-8. 789 109. Ryter SW, Alam J, Choi AM. Heme oxygenase-1/carbon monoxide: from basic science to 790 therapeutic applications. Physiol Rev. 2006;86(2):583-650. 791 110. Schillinger M, Exner M, Mlekusch W, Domanovits H, Huber K, Mannhalter C, et al. Heme 792 oxygenase-1 gene promoter polymorphism is associated with abdominal aortic aneurysm. Thromb 793 Res. 2002;106(2):131-6. 794 111. Gregorek AC, Gornik KC, Polancec DS, Dabelic S. GT microsatellite repeats in the heme 795 oxygenase-1 gene promoter associated with abdominal aortic aneurysm in Croatian patients. 796 Biochem Genet. 2013;51(5-6):482-92. 797 112. Yamada N, Yamaya M, Okinaga S, Nakayama K, Sekizawa K, Shibahara S, et al. Microsatellite 798 polymorphism in the heme oxygenase-1 gene promoter is associated with susceptibility to 799 emphysema. Am J Hum Genet. 2000;66(1):187-95. 800 113. Nishiyama A, Ohno T, Iwata S, Matsui M, Hirota K, Masutani H, et al. Demonstration of the 801 interaction of thioredoxin with p40phox, a phagocyte oxidase component, using a yeast two-hybrid 802 system. Immunol Lett. 1999;68(1):155-9. 803

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114. Billiet L, Furman C, Larigauderie G, Copin C, Brand K, Fruchart JC, et al. Extracellular human 804 thioredoxin-1 inhibits lipopolysaccharide-induced interleukin-1beta expression in human monocyte-805 derived macrophages. The Journal of biological chemistry. 2005;280(48):40310-8. 806 115. Tamaki H, Nakamura H, Nishio A, Nakase H, Ueno S, Uza N, et al. Human thioredoxin-1 807 ameliorates experimental murine colitis in association with suppressed macrophage inhibitory factor 808 production. Gastroenterology. 2006;131(4):1110-21. 809 116. Yoshida M, Korfhagen TR, Whitsett JA. Surfactant protein D regulates NF-kappa B and matrix 810 metalloproteinase production in alveolar macrophages via oxidant-sensitive pathways. J Immunol. 811 2001;166(12):7514-9. 812 117. Yang H, Roberts LJ, Shi MJ, Zhou LC, Ballard BR, Richardson A, et al. Retardation of 813 atherosclerosis by overexpression of catalase or both Cu/Zn-superoxide dismutase and catalase in 814 mice lacking apolipoprotein E. Circulation research. 2004;95(11):1075-81. 815 118. Ramos-Mozo P, Madrigal-Matute J, Martinez-Pinna R, Blanco-Colio LM, Lopez JA, Camafeita 816 E, et al. Proteomic analysis of polymorphonuclear neutrophils identifies catalase as a novel 817 biomarker of abdominal aortic aneurysm: potential implication of oxidative stress in abdominal 818 aortic aneurysm progression. Arterioscler Thromb Vasc Biol. 2011;31(12):3011-9. 819 119. Parastatidis I, Weiss D, Joseph G, Taylor WR. Overexpression of catalase in vascular smooth 820 muscle cells prevents the formation of abdominal aortic aneurysms. Arterioscler Thromb Vasc Biol. 821 2013;33(10):2389-96. 822 120. Sinha I, Pearce CG, Cho BS, Hannawa KK, Roelofs KJ, Stanley JC, et al. Differential regulation 823 of the superoxide dismutase family in experimental aortic aneurysms and rat aortic explants. J Surg 824 Res. 2007;138(2):156-62. 825 121. Hao Q, Chen X, Wang X, Dong B, Yang C. Curcumin Attenuates Angiotensin II-Induced 826 Abdominal Aortic Aneurysm by Inhibition of Inflammatory Response and ERK Signaling Pathways. 827 Evid Based Complement Alternat Med. 2014;2014:270930. 828 122. Piechota-Polanczyk A, Goraca A, Demyanets S, Mittlboeck M, Domenig C, Neumayer C, et al. 829 Simvastatin decreases free radicals formation in the human abdominal aortic aneurysm wall via NF-830 kappaB. Eur J Vasc Endovasc Surg. 2012;44(2):133-7. 831 123. Watanabe A, Ichiki T, Sankoda C, Takahara Y, Ikeda J, Inoue E, et al. Suppression of 832 abdominal aortic aneurysm formation by inhibition of prolyl hydroxylase domain protein through 833 attenuation of inflammation and extracellular matrix disruption. Clin Sci (Lond). 2014;126(9):671-8. 834 124. Grigoryants V, Hannawa KK, Pearce CG, Sinha I, Roelofs KJ, Ailawadi G, et al. Tamoxifen up-835 regulates catalase production, inhibits vessel wall neutrophil infiltration, and attenuates 836 development of experimental abdominal aortic aneurysms. Journal of vascular surgery. 837 2005;41(1):108-14. 838 125. Smith JV, Luo Y. Studies on molecular mechanisms of Ginkgo biloba extract. Appl Microbiol 839 Biotechnol. 2004;64(4):465-72. 840 126. Wang L, Bai Y, Wang B, Cui H, Wu H, Lv JR, et al. Suppression of experimental abdominal 841 aortic aneurysms in the mice by treatment with Ginkgo biloba extract (EGb 761). J Ethnopharmacol. 842 2013;150(1):308-15. 843 127. Goel A, Kunnumakkara AB, Aggarwal BB. Curcumin as "Curecumin": from kitchen to clinic. 844 Biochem Pharmacol. 2008;75(4):787-809. 845 128. Fan J, Li X, Yan YW, Tian XH, Hou WJ, Tong H, et al. Curcumin attenuates rat thoracic aortic 846 aneurysm formation by inhibition of the c-Jun N-terminal kinase pathway and apoptosis. Nutrition. 847 2012;28(10):1068-74. 848 129. Parodi FE, Mao D, Ennis TL, Pagano MB, Thompson RW. Oral administration of 849 diferuloylmethane (curcumin) suppresses proinflammatory cytokines and destructive connective 850 tissue remodeling in experimental abdominal aortic aneurysms. Ann Vasc Surg. 2006;20(3):360-8. 851 130. Fong GH, Takeda K. Role and regulation of prolyl hydroxylase domain proteins. Cell Death 852 Differ. 2008;15(4):635-41. 853

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131. Dubey RK, Tyurina YY, Tyurin VA, Gillespie DG, Branch RA, Jackson EK, et al. Estrogen and 854 tamoxifen metabolites protect smooth muscle cell membrane phospholipids against peroxidation 855 and inhibit cell growth. Circulation research. 1999;84(2):229-39. 856 132. Maiellaro-Rafferty K, Weiss D, Joseph G, Wan W, Gleason RL, Taylor WR. Catalase 857 overexpression in aortic smooth muscle prevents pathological mechanical changes underlying 858 abdominal aortic aneurysm formation. Am J Physiol Heart Circ Physiol. 2011;301(2):H355-62. 859 133. Singh U, Devaraj S, Jialal I. Vitamin E, oxidative stress, and inflammation. Annu Rev Nutr. 860 2005;25:151-74. 861 134. Moat SJ, Clarke ZL, Madhavan AK, Lewis MJ, Lang D. Folic acid reverses endothelial 862 dysfunction induced by inhibition of tetrahydrobiopterin biosynthesis. Eur J Pharmacol. 863 2006;530(3):250-8. 864 135. Verhaar MC, Stroes E, Rabelink TJ. Folates and cardiovascular disease. Arterioscler Thromb 865 Vasc Biol. 2002;22(1):6-13. 866 136. Daugherty A, Cassis LA. Mouse models of abdominal aortic aneurysms. Arterioscler Thromb 867 Vasc Biol. 2004;24(3):429-34. 868 137. Gopal K, Nagarajan P, Jedy J, Raj AT, Gnanaselvi SK, Jahan P, et al. beta-Carotene Attenuates 869 Angiotensin II-Induced Aortic Aneurysm by Alleviating Macrophage Recruitment in Mice. PLoS One. 870 2013;8(6):e67098. 871 138. Tornwall ME, Virtamo J, Haukka JK, Albanes D, Huttunen JK. Alpha-tocopherol (vitamin E) 872 and beta-carotene supplementation does not affect the risk for large abdominal aortic aneurysm in a 873 controlled trial. Atherosclerosis. 2001;157(1):167-73. 874 139. May JM. How does ascorbic acid prevent endothelial dysfunction? Free Radic Biol Med. 875 2000;28(9):1421-9. 876 140. Tanaka A, Hasegawa T, Morimoto K, Bao W, Yu J, Okita Y, et al. Controlled release of ascorbic 877 acid from gelatin hydrogel attenuates abdominal aortic aneurysm formation in rat experimental 878 abdominal aortic aneurysm model. Journal of vascular surgery. 2014;60(3):749-58. 879 141. Frankel EN, Waterhouse AL, Kinsella JE. Inhibition of human LDL oxidation by resveratrol. 880 Lancet. 1993;341(8852):1103-4. 881 142. Martinez J, Moreno JJ. Effect of resveratrol, a natural polyphenolic compound, on reactive 882 oxygen species and prostaglandin production. Biochem Pharmacol. 2000;59(7):865-70. 883 143. Orallo F, Alvarez E, Camina M, Leiro JM, Gomez E, Fernandez P. The possible implication of 884 trans-Resveratrol in the cardioprotective effects of long-term moderate wine consumption. Mol 885 Pharmacol. 2002;61(2):294-302. 886 144. Csiszar A, Labinskyy N, Podlutsky A, Kaminski PM, Wolin MS, Zhang C, et al. Vasoprotective 887 effects of resveratrol and SIRT1: attenuation of cigarette smoke-induced oxidative stress and 888 proinflammatory phenotypic alterations. Am J Physiol Heart Circ Physiol. 2008;294(6):H2721-35. 889 145. Norata GD, Marchesi P, Passamonti S, Pirillo A, Violi F, Catapano AL. Anti-inflammatory and 890 anti-atherogenic effects of cathechin, caffeic acid and trans-resveratrol in apolipoprotein E deficient 891 mice. Atherosclerosis. 2007;191(2):265-71. 892 146. Brakenhielm E, Cao R, Cao Y. Suppression of angiogenesis, tumor growth, and wound healing 893 by resveratrol, a natural compound in red wine and grapes. FASEB J. 2001;15(10):1798-800. 894 147. Palmieri D, Pane B, Barisione C, Spinella G, Garibaldi S, Ghigliotti G, et al. Resveratrol 895 counteracts systemic and local inflammation involved in early abdominal aortic aneurysm 896 development. J Surg Res. 2011;171(2):e237-46. 897 148. Perez-Vizcaino F, Duarte J, Andriantsitohaina R. Endothelial function and cardiovascular 898 disease: effects of quercetin and wine polyphenols. Free Radic Res. 2006;40(10):1054-65. 899 149. Wang L, Wang B, Li H, Lu H, Qiu F, Xiong L, et al. Quercetin, a flavonoid with anti-900 inflammatory activity, suppresses the development of abdominal aortic aneurysms in mice. Eur J 901 Pharmacol. 2012;690(1-3):133-41. 902

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150. Wang L, Cheng X, Li H, Qiu F, Yang N, Wang B, et al. Quercetin reduces oxidative stress and 903 inhibits activation of cJun Nterminal kinase/activator protein1 signaling in an experimental mouse 904 model of abdominal aortic aneurysm. Mol Med Rep. 2014;9(2):435-42. 905 151. Golledge J, Norman PE. Current status of medical management for abdominal aortic 906 aneurysm. Atherosclerosis. 2011;217(1):57-63. 907 152. Meijer CA, Stijnen T, Wasser MN, Hamming JF, van Bockel JH, Lindeman JH. Doxycycline for 908 stabilization of abdominal aortic aneurysms: a randomized trial. Ann Intern Med. 2013;159(12):815-909 23. 910 153. Sillesen H, Eldrup N, Hultgren R, Lindeman J, Bredahl K, Thompson M, et al. Randomized 911 clinical trial of mast cell inhibition in patients with a medium-sized abdominal aortic aneurysm. Br J 912 Surg. 2015;102(8):894-901. 913

914

915

916

917

918

919

920

921

922

923

924

925

926

927

928

929

930

931

932

933

934

935

24

936

937

938

Figure legends 939

Figure 1 A Schematic diagram showing the critical balance between oxidants and antioxidants 940

in relation to AAA [modified from [(1)]. 941

Endogenous oxidant systems generate reactive oxygen species (ROS) whilst the antioxidants either 942

detoxify or scavenge ROS. A shift in the balance towards increased oxidant activity, leads to excess 943

ROS and resultant oxidative stress. Oxidative stress with associated tissue damage and other pro-944

aneurysmal factors may result in AAA. 945

Abbreviations: *may generate ROS, AAA= abdominal aortic aneurysm, COX= cyclooxygenase, 946

ECM= extracellular matrix, LO= lipoxygenase, HO= heme oxygenase, NOS= nitric oxide synthase, 947

NOX= nicotinamide adenine dinucleotide phosphate oxidase, ROS= reactive oxygen species, SOD= 948

superoxide dismutase, TRX= thioredoxin, VSMC= vascular smooth muscle cells, XO= xanthine 949

oxidase. 950

951

Figure 2 Overview of the mechanism leading to oxidative stress and potential treatments to limit 952

oxidative stress associated with AAA. 953

AAA risk factors include smoking, male gender, advanced age and family history. Pro-inflammatory 954

agents include Ang II. ROS generating enzymes include eNOS, NOX, XO, LO, COX. ROS degrading 955

systems include SOD and catalase. O2 is converted to O2− by NOX. SOD inactivates O2− forming 956

H2O2, and catalase converts H2O2 to H2O and O2. Under pro-aneurysmal conditions, H2O2 could be 957

converted to •OH by the Fenton and/or the Haber-Weiss reactions. In addition, O2− then combines 958

with NO produced by eNOS to form the highly reactive ONOO-. The increase generation of ROS 959

within the aorta results in oxidative stress which leads to enhance ACE expression which promotes 960

Ang II production and resultant positive feedback activation of more pro-oxidant enzymes. Oxidative 961

stress also promotes mitochondria dysfunction, activates signalling molecules, and potentiates 962

inflammation. It also increases pro-inflammatory cytokine secretion with resultant recruitment of 963

effector immune cells, release of proteases, leading to ECM degradation, VSMC apoptosis and 964

consequent AAA development and progression. Key targets currently under investigation include: 1). 965

Preventing eNOS uncoupling using vitamins, 2). Preventing the production of O2− using NOX-966

inhibitors, TRX or genetic approaches, 3). Blocking the COX-2 and/or LO pathways using vitamins 967

and NSAID, 4). Enhancing the effect of SOD using SOD mimetics, folic acid and polyphenolics, 5). 968

Applying catalase activators to improve the detoxification of H2O2, 6). Using polyphenolics to inhibit 969

ROS-induced signalling and 7). Employing vitamins, polyphenolics and NSAID to scavenge ROS and 970

prevent oxidative stress. 971

Abbreviations: 5-LO= 5-lipooxygenase, AAA= abdominal aortic aneurysm, ACE= angiotensin 972

converting enzyme, Ang I= angiotensin I, Ang II= angiotensin II, AP-1= activator protein-1, COX= 973

cyclooxygenase, ECM= extracellular matrix, eNOS= endothelial nitric oxide synthase, Erk1/2= 974

extracellular signal-regulated kinase 1/2, Fe2+= ferrous iron, Fe3+= ferric iron, H2O2= hydrogen 975

peroxide, IL-1β= interleukin-1 beta, JNK= c‑Jun N‑terminal kinase, LO= lipoxygenase, MCP-1= 976

25

monocyte chemoattractant protein-1, MMP= matrix metalloproteinase, NF-κβ= nuclear factor kappa 977

beta, NO= nitric oxide, NOS= nitric oxide synthase, NSAIDS= nonsteroidal anti-inflammatory drugs, 978

NOX= nicotinamide adenine dinucleotide phosphate oxidase, O2-= superoxide, •OH= hydroxyl 979

radical, ONOO- = peroxynitrite, ROS= reactive oxygen species, SOD= superoxide dismutase, TNF-980

α= tumour necrosis factor-α, VSMC= vascular smooth muscle cells, XO= xanthine oxidase. Symbols 981

indicate: ↑increase; ↓decrease. 982

983

984

985

986

987

988

989

990

991

992

993

994

995

996

997

998

999

1000

1001

1002

1003

1004

1005

1006

26

1007

1008

1009

Tables 1010

Table 1 Overview of studies assessing the lipoxygenase pathway in AAA 1011

Therapies Key findings AAA model Reference

BLT1 inhibition

(genetic).

Inhibition of the 5-LO metabolite, LTB4

mediated-inflammation. Decreased MMP-2

and -9 expression. Decreased inflammatory

cell infiltrate and decreased AAA

formation.

Ang II-infused

ApoE–/– mouse

model of AAA.

(72)

5-LO inhibition

(genetic and

pharmacological).

Decreased MMP-9 activity and effector

immune cell recruitment. 71% (genetic)

and 54% (pharmacological) reduction in

aortic dilatation.

Elastase perfused-

and Ang II-infused

Ldlr-/- mouse

models of AAA.

(67)

None. Upregulated 5-LO, FLAP, and LTC4S

transcripts in aneurysmal aorta compared

with non-aneurysmal control aortas.

Human. (74)

5-LO inhibition

genetic deletion).

Decreased MMP-2 and MIP-1α expression.

Decreased AAA incidence.

Cholate fed ApoE–/–

mice.

(66)

BLT1 inhibition

(pharmacological)

.

Diminished macrophage accumulation,

decreased incidence of AAA when

administered at the same time as Ang II,

but no significant effect on AAA size when

administered 14 days after AAA induction.

Ang II-infused

ApoE–/– mouse

model of AAA.

(68)

Generalised ROS

inhibition

(endaravone).

Decreased MMP-2, MMP-9, and ROS

expression. Reduced AAA formation

(when administered from the onset of AAA

induction), and expansion (when

administered to already established AAAs).

Combined elastase-

induced and CaCl2-

induced rat models

of AAA.

(71)

5-LO inhibition

(genetic and

pharmacological).

No significant effect on aortic media

inflammation or AAA development in 5-

LO deficient mice or mice administered

FLAP inhibitor (MK-0591).

Ang II-infused

ApoE–/– mouse

model of AAA.

(73)

None. No association between 7 single nucleotide

polymorphisms in ALOX5AP, the gene

encoding FLAP with human AAA.

Human. (76)

27

Abbreviations: 5-LO= 5-lipooxygenase, Ang II= angiotensin-II, ApoE–/– = apolipoprotein E-deficient, 1012

BLT1= leukotriene B4 receptor-1, FLAP= 5-LO activating protein, CaCl2= calcium chloride, Ldlr-/- = 1013

low density lipoprotein receptor knockout, LTB4= leukotriene B4, LTC4S= leukotriene C4 synthase, 1014

MIP= macrophage inflammatory protein, MMP== matrix metalloproteinase, ROS= reactive oxygen 1015

species. 1016

Table 2 Studies assessing the association of the cyclooxygenase pathway with AAA 1017

Therapies Key findings AAA model Reference

COX-2 inhibition

(pharmacological).

Decreased incidence and

severity of AAA.

Ang II-infused

mouse model of

AAA.

(82) (84)

(85)

COX-2 inhibition

(genetic deletion).

Decreased MCP-1, MIP-1α

expression. Decreased

macrophage infiltration.

Decreased AAA formation.

Ang II-infused

mouse model of

AAA.

(81)

COX-2 inhibition

(genetic deletion).

No significant effect on AAA

incidence.

Ang II-infused

ApoE–/– mouse

model of AAA.

(83)

COX-2 inhibition

(pharmacological).

Decreased MMP-9 expression

and decreased AAA

development.

Decreased PGE2 and MMP-9

expression. Decreased

inflammatory cell infiltration.

Decreased AAA expansion.

Elastase-induced rat

model of AAA.

(86)

(87) (88)

COX-2 inhibition

(pharmacological).

Decreased MMP-9 expression

but no significant effect on

AAA expansion.

Elastase-induced rat

model of AAA.

(89)

None Significant reduction in AAA

growth in patients on NSAIDS

compared to patients without

NSAIDS.

Human case-control

study.

(80)

1018

Abbreviations: Ang II= angiotensin-II, ApoE–/– = apolipoprotein E-deficient, COX-2= 1019 cyclooxygenase-2, MCP= monocyte chemoattractant protein, MIP= macrophage inflammatory 1020 protein, MMP= matrix metalloproteinase, NSAIDS= nonsteroidal anti-inflammatory drugs, PGE2= 1021 prostaglandin E-2, VSMC= vascular smooth muscle cell. 1022

1023

1024

1025

1026

28

1027

1028

1029

Table 3 Overview of studies assessing the effects of medications known to affect NOS and/or direct 1030

NOS inhibition in AAA 1031

Therapies Key findings AAA model Reference

Simvastatin Decreased MMP-9, MMP-3, and

NF-κβ expression. Increased

TIMP-1 expression. Decreased

AAA formation.

Elastase-induced

mouse model of

AAA.

(147)

(Steinmetz

2005)

Simvastatin Decreased macrophage

infiltration. No significant effect

on AAA diameter.

Ang II-infused

ApoE–/– and Ldlr-/-

mouse models of

AAA.

(153)

(Golledge

2010)

Fenofibrate Decreased inflammatory cell and

cytokine expression. Decreased

VSMC apoptosis. Decreased

AAA development.

Ang II-infused

ApoE–/– and Ldlr-/-

mouse models of

AAA.

(105)

(120)

iNOS inhibition

(genetic).

Decreased MMP-9, MMP-2, and

NO expression. Decreased AAA

formation.

CaCl2-induced

mouse model of

AAA.

(18)

H4B and eNOS

inhibition (genetic).

Increased MMP-9, MMP-2

expression. Increased

macrophage infiltration.

Increased AAA formation. Folic

acid administration blocked the

AAA promoting effect of H4B

deficiency.

Ang II-infused

mouse model of

AAA.

(94)

eNOS upregulation

(pharmacological).

Decreased ECM degradation.

Decreased macrophage

infiltration. Decreased O2−

production. Decreased AAA

expansion.

Ang II-infused

mouse model of

AAA.

(95)

1032

Abbreviations: Ang II= angiotensin-II, ApoE–/– = apolipoprotein E-deficient, CaCl2= calcium 1033

chloride, ECM= extracellular matrix, eNOS= endothelial nitric oxide synthase, H4B= 1034

tetrahydrobiopterin, iNOS= inducible nitric oxide synthase, Ldlr-/- = low density lipoprotein receptor 1035

knockout, MMP= matrix metalloproteinase, NF-κβ= nuclear factor kappa beta, NO= nitric oxide, 1036

29

O2−= superoxide , TIMP-1= tissue inhibitor of matrix metellaoproteinase-1, VSMC= vascular smooth 1037

muscle cells. 1038

1039

1040

Table 4 Overview of studies assessing superoxide dismutase and/or catalase in AAA 1041

Therapies Key findings AAA model Reference

SOD inhibition

(pharmacological).

Decreased MMP-2,

MMP-9, and NF-κβ

expression. Decreased

SOD and catalase

activity. Decreased AAA

size.

CaCl2-induced mouse

model of AAA.

(126)

SOD upregulation

(pharmacological).

Decreased aortic MCP-1,

and TNF-α expression.

Decreased macrophage

infiltration. Increased

SOD activity. Decreased

AAA incidence.

Ang II-infused ApoE–/–

mouse model of AAA.

(121)

None. Higher catalase activity.

Reduced NF-κβ

expression. No effect on

SOD activity and H2O2

in AAA wall in vitro.

Human. (122)

Catalase upregulation

(pharmacological).

Decreased aortic

neutrophil infiltration.

Decreased AAA

expansion in rats

receiving tamoxifen to

increase catalase

expression. A catalase

inhibitor (AT) blocked

the AAA protective

effects of tamoxifen.

Elastase-induced rat

model of AAA.

(124)

Catalase upregulation

(genetic).

Transgenic mice

overexpressing catalase

exhibited increased H2O2 degradation and

decreased aortic wall

remodelling.

Ang II-infused mouse

model of AAA.

(163)

(Maiellaro_rafferty,

2011)

Catalase upregulation

(genetic and

pharmacological).

Decreased MMP activity.

Decreased VSMC

apoptosis. Decreased

AAA formation.

CaCl2-induced mouse

model of AAA.

(119)

Abbreviations: Ang II= angiotensin-II, ApoE–/– = apolipoprotein E-deficient, AT=3-amino-1, 2, 4-1042 triazole, CaCl2= calcium chloride, H2O2= hydrogen peroxide, MMP== matrix metalloproteinase, 1043 MnSOD= manganese sup